比较3种形态学方法观察成骨细胞矿化结节的应用价值

Comparison of three morphology methods for observing mineralization nodules of osteoblasts

背景:矿化结节是成骨细胞分化成熟的标志,以往观察方法多采用茜素红等特殊染色法。目的:比较茜素红染色-光镜、四环素荧光标记-激光扫描共聚焦显微镜、扫描电镜3种形态学观察成骨细胞矿化结节的方法,分析各自的特点及在骨病研究中的应用价值。方法:将大鼠成骨细胞株UMR-106正常培养、每天换液,连续培养14 d,分别采用茜素红染色-光镜、四环素荧光标记-激光扫描共聚焦显微镜、扫描电镜观察矿化结节的形态结构,并利用扫描电镜结合能谱仪原位定量分析钙元素;此外,在培养中加入可抑制成骨细胞增殖、分化的肿瘤坏死因子α作为对照实验。结果与结论:3种观察方法均可观察到正常成骨细胞矿化结节的形态结构。对于肿瘤坏死因子α抑制成骨细胞产生矿化结节的情况,经茜素红染色-光镜观察法,未见明显的矿化结节;而四环素荧光染色-激光扫描共聚焦显微镜观察法,可见清晰、稀少的矿化结节;扫描电镜观察法明显可见较少而细小的矿化结节,提示后二种方法灵敏度较高。此外,扫描电镜可将观察成骨细胞分泌钙质、形成矿化结节的亚细胞结构,与能谱元素分析结合,可实现矿化结节的定位与定量,在骨病研究中值得推广应用。

BACKGROUND：Mineralized nodules are the mature marker of osteoblast differentiation, and the observation methods mainly use alizarin red staining. OBJECTIVE：To compare the observation results of mineralized nodules by three methods, and to explore their characteristics and advantages, as wel as further application in the research of bone disease. METHODS：The rat osteoblast-like cellline UMR-106 were cultured in the fresh medium that was changed every day, for 14 days. Alizarin red staining-light microscope, tetracycline fluorescence labeling-laser confocal scanning microscopy, and scanning electron microscopy were used to observe mineralized nodules. The calcium content of mineralized nodules was quantified using scanning electron microscopy and energy dispersive spectroscopy. In addition, tumor necrosis factor alpha that could inhibit the proliferation and differentiation of osteoblasts was used as the control. RESULTS AND CONCLUSION：The three morphology methods could be used to observe the mineralized nodules of normal osteoblasts. As for tumor necrosis factor alpha, no mineralized nodules of osteoblasts were observed by alizarin red staining-light microscopy;smal mineralized nodules were observed by tetracycline staining-laser scanning confocal microscopy and scanning electron microscopy, suggesting tetracycline staining and scanning electron microscopy were more sensitive in the observation. Scanning electron microscopy could be used to observe the submicroscopic structures of mineralized nodules in the osteoblasts, and the formation of mineralized nodules, including the calcium secretion. Additional y, scanning electron microscopy combined with energy dispersive spectroscopy analysis can successful y quantify and position the mineralized nodules, indicating a potential application in the research of bone diseases.